High concentrations of readily chelatable (mobile) Zn2+ are found in various brain regions, yet the details of zinc signaling pathways in areas such as CNS development, learning and memory formation, and motor and sensory function, remain elusive. These dynamic pools of mobile Zn2+ may be tracked using fluorescent zinc sensors and are the focus of many studies aimed at understanding the function of zinc- enriched neurons; however, few have attempted to elucidate the molecular mechanisms of Zn2+ during neurogenesis or clarify zinc signaling pathways in motor and sensory functions. To address these deficiencies, we propose the development of a category of zinc-selective sensors that combine the advantages of tunable small molecule sensors and genetically encodable tags. We will design, prepare, and characterize reaction-based small molecule-protein hybrid fluorescent zinc sensors for targeting subcellular organelles and cell membranes. Specifically, we will prepare a reaction-based hybrid green fluorescent zinc sensor for targeting the mitochondria, nucleus, cytosol, and endoplasmic reticulum in proliferating and differentiated neural progenitor cells (NPCs) and use it to map mobile Zn2+ pools in NPCs at discrete locales. Hybrid probes will be characterized in vitro using purified proteins and functionalized small molecule sensors, protein tag-expressing genes cloned to include subcellular targeting sequences, and in cellulo imaging carried out to monitor sensor localization and relative endogenous zinc levels. We will also develop a red-emitting reaction-based hybrid probe for quantifying mobile zinc in NPCs. This will be approached through the design, preparation, and characterization of a protein tag fused to a fluorescent protein, followed by in cellulo fluorescence microscopy for calibration and then endogenous Zn2+ quantification. Finally, we will develop a novel cell-surface displayed hybrid fusion protein for probing synaptic zinc because imaging zinc transmission with high spatiotemporal resolution requires novel sensors that can be localized to neuronal cell surfaces. We will employ a transmembrane protein fused to a protein tag that can be coupled with an extracellular zinc sensor for imaging in cell culture, and with collaborators, in brain tissue slices of relevance to auditory function. This study will be of great importance to the fields of bioinorganic chemistry and neuroscience by providing novel sensors that will be utilized to directly probe mobile zinc signaling pathways in various neurobiological platforms and which can then be built upon to address various broader biological questions.